| Troxerutin is a semi synthetic flavonoids rutin made by hydroxyethylation of rutin.It is the main component of Venoruton,a cardiovascular drugs,and it used to treat cerebral infarction,apoplexy sequela and other diseases in clinic.Rutin used for prevention and treatment of cerebral haemorrhage,hypertension,retinal hemorrhage,purpura and acute hemorrhagic nephritis and other diseases in clinic,treatment of cerebral vascular disease with 87.8% efficiency.Troxerutin aglycone is obtained by hydrolyzing troxerutin.In the structure,troxerutin is more than three hydroxyethyl compare with rutin and more than two rutinose compare with troxerutin aglycone.There are reports of flavonoid compounds is mainly the aglycones,hydrolyzed by glycosidic bond,exert pharmacological effects in the organism.In recent years,several articles about the brain pharmacological effect of troxerutin are reported.This research mainly study on the physicochemical properties,the brain pharmacodynamics,the brain pharmacokinetics and the part mechanism of troxerutin and analogues through the blood-brain barrier.Mainly as follows:Part one: Physicochemical properties of Troxerutin and analoguesThe equilibrium solubility in water,ethanol and the oil-water partition coefficient in octanol-water of troxerutin and analogues is determined by using the flask-UPLC method.Under the temperature of 37?,equilibrium solubility of troxerutin,rutin and troxerutin aglycone is respectively>1000 g·L-1,0.16±0.01 g·L-1,0.12±0.01g·L-1in water,2.13±0.02 g·L-1,3.34±0.09 g·L-1,1.53±0.06g·L-1in ethanol.Showed that the fat soluble of troxerutin,rutin,troxerutin aglycone is increasing;the log P value of troxerutin,rutin,troxerutin aglycone were-1.9195±0.10?-0.7308±0.20?1.7922±0.10.Preliminary deduce that the ability of through the blood-brain barrier of troxerutin,rutin,troxerutin aglycone maybe gradually increased.Part two: Effect of Troxerutin and analogues on learning and memoryIn order to investigate cerebral pharmacological activity of troxerutin and analogues,60 male SD rats were divided into six groups: positive drug group,troxerutin group,rutin group,troxerutin glycosides group,model group and blank group,ten rats in each group.Positive group were intraperitoneally injected with 1.3g·kg-1piracetam,drug group were intraperitoneally injected 52 mg·kg-1troxerutin,rutin or troxerutin aglycone,model group and blank group were intraperitoneally injected with the same volume of physiological saline.Establishing the model of learning and memory impairment by intraperitoneal injection of 1.5 mg·kg-1scopolamine after administration for ten days.Using Morris water maze and Y maze to investigate the effect of troxerutin and analogues on learning and memory impairment caused by scopolamine.The results showed that: troxerutin and rutin can significantly improve the learning and memory impairment caused by scopolamine?P < 0.05?,troxerutin aglycone has no obvious effect?P > 0.05?.Part three: Brain pharmacokinetics of troxerutin and analoguesEstablishing the UPLC-MS/MS analytical method for simultaneous determination of troxerutin and analogues in mice brain tissue by using methanol protein precipitation toextracte troxerutin,rutin and troxerutin aglycone.The method was proves to be linear from 1400ng·ml-1for troxerutin,rutin and troxerutin aglycone in mice brain tissue,the matrix effect were ranged from 99% 100%,the recovery rate were ranged from92% 99%,the intra-day and inter-day precision values were both below 6.0%,the stability were higher than 5.0%.The method is proved to be simple,sensitive,accurate and suitable for the pharmacokinetic study of troxerutin and analogues in mice brain tissue.SD mice were caudal vein injection of troxerutin and analogues for 60mg·kg-1and the brain tissues was collected at 0.08 h,0.25 h,0.5h,1h,2h,4h,6h,8h,12 h,24h and analyzed by UPLC-MS/MS.Calculating the pharmacokinetic parameters with the atrioventricular model,and the main pharmacokinetic parameters of troxerutin,rutin and troxerutin aglycone were as follows: Tmax were all 0.8min,Cmax were 888.07±15.06ng·g-1,303.20±5.73ng·g-1and 294.72±10.00ng·g-1,t1/2 were 6.01±1.39 h,6.86±2.34 h and 5.29±1.25 h,MRT were 2.43±0.10 h,2.29±0.19 h and 2.73±0.17 h,K were 0.11 ± 0.03h-1,0.11 ± 0.04h-1and 0.14 ± 0.03h-1,AUC0-t were 495.64 ±14.36ng·g-1·h,180.08±6.09ng·g-1·h and 193.65±9.77ng·g-1·h.And Cmax and AUC0-t of troxerutin group was higher than the other two groups.It indicated that troxerutin,the molecular weight maximum and log P minimum,through blood-brain barrier with maximum amount,and suggest that there may be other factors involved in troxerutin and analogues through blood-brain barrier.Part four: The part mechanism of troxerutin and analogues throughing blood-brain barrierEstablishing the high expression of P-gp in MDCK cells through induced by 1?mol·L-1dexamethasone.Then the MDCK cell membrane model was used to investigate the transport of troxerutin and analogues with 200?mol·L-1and determined by UPLC-MS/MS.Calculating the apparent permeability coefficient?Papp?and efflux rate?ER?and investigating influence by P-gp inhibitors on their transport.The results showed: dexamethasone can upregulate the function of P-gp by significantly promote the rhodamine-123 efflux in MDCK cells,it can be used to establish the high expression of P-gp MDCK cell membrane;In the transport experiment,the Papp of troxerutin,rutin and troxerutin aglycone from AP to BL were?1.49±0.10?×10-7cm·s-1,?1.35±0.08?×10-7cm·s-1and?1.09±0.07?×10-7cm·s-1,from BL to AP were?1.82±0.07?× 10-7cm·s-1,?1.77 ± 0.08?× 10-7cm·s-1and?1.55±0.13?×10-7cm·s-1,ER were 1.22±0.05,1.32±0.09 and 1.42±0.06,after using P-gp inhibitor Verapamil,the Papp of troxerutin,rutin and troxerutin aglycone from AP to BL increased significantly?P<0.01?and the Papp were?1.97±0.11?×10-7cm·s-1,?1.76±0.09?×10-7cm·s-1and?1.54±0.04?×10-7cm·s-1,and Papp from BL to AP were?1.79±0.07?×10-7cm·s-1,?1.66±0.07?×10-7cm·s-1and?1.40±0.10?×10-7cm·s-1with decreasing trend,ER were reduced to 0.91±0.05,0.94±0.06 and 0.91±0.06 with significant decreasing trend?P<0.01?,and ER of troxerutin aglycone was significant bigger than ER of troxerutin?P<0.01?.Suggesting that P-gp may be involved in the transport of troxerutin and analogues throughing blood-brain barrier and may have large effect to troxerutin aglycone,and it may be the most important reason that troxerutin aglycone,the lightest molecule weight and the biggest fat-soluble,trough the blood-brain barrier least.Although,the mechanism of troxerutin and analogues through the blood-brain barrier may also relate to other receptor. |
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